Hereditary lack of adenosine deaminase (ADA) is the most common cause of severe combined immunodeficiency disease, a lethal childhood disorder. Cells from affected children have less than 1% ADA deficiency. There are 3 long-term objectives: 1) to clone, map, and sequence the normal human ADA gene, 2) to compare nucleotide sequences and patterns of organization of mutant ADA genes from ADA deficient children with the normal, and 3) to explain cellular ADA deficiency in molecular terms which link changes in the structure and expression of the ADA gene to changes in structure and/or amounts of ADA mRNA and protein. Cell lines are available from 19 different ADA deficient children. The lines express the enzyme deficiency in vitro and represent readily available soures of mutant ADA proteins, mRNAs, and genomic DNAs. Studies of the mutant ADA proteins and mRNAs have proved that the lines represent a large number of different mutations in the ADA gene. The full length cDNA to normal human ADA mRNA has been cloned and sequenced. Normal and selected mutant ADA genes will be cloned in phage vectors and will be identified by hybridization to the ADA cDNA probe. The normal ADA gene will be sequenced by the Sanger procedure and the organization of exons, introns, and flanking sequences will be defined. A set of restriction fragments representing the separate introns, exons, and flanking regions will be prepared and cloned. These will serve as probes for sequences representing different regions of the normal gene and will be useful reagents for characterization of mRNA precursors and mutant genes. ADA mRNAs from ADA deficient cells will be quantitated by dot blot, sized by Northern gel electrophoresis, and mapped by the S1 nuclease technique. Using defects identified in mRNAs as a guide to probable sites of mutation, appropriate regions of cloned mutant genomic DNAs or cloned cDNAs will be sequenced and compared to normal. A variety of techniques including in vitro transcription, translation, and transfection will provide additional functional characterization of normal and mutant ADA genes. There is a high probability that these studies will identify the molecular defects in a series of mutant alleles responsible for a fatal hereditary disease. The information is also relevant to the prenatal diagnosis of ADA deficiency and to the design of procedures for transfer of normal ADA genes to defective cells.